人胰岛素基因在乳酸菌中的表达及其对非肥胖糖尿病(NOD)小鼠的作用

为实现人胰岛素基因在乳酸菌中的表达及探索其用作口服疫苗治疗Ⅰ型糖尿病(T1DM)的可行性,首先将人胰岛素基因密码子替换为乳酸菌偏爱密码子,同时在A,B链序列间加入连接短肽序列,经引物退火拼接合成人胰岛素基因。克隆至乳酸菌表达载体中后,利用电击转化法实现了带信号肽SPUsp45的人胰岛素基因在乳酸乳球菌(Lactococcus lactis)MG1363和干酪乳杆菌(Lactobacillus casei)ATCC27092中的表达。Western blot检测显示重组胰岛素位于细胞壁上,当菌体生长到OD600为0.4时达到最大表达量。用含有表达重组人胰岛素的Lactobacillus caseiATCC27092/pSW501菌体饲喂非肥胖糖尿病(NOD)小鼠,发现可刺激小鼠产生特异性抗体,同时使与免疫耐受相关的细胞因子IL-4水平明显升高(38.583±2.083pg/mL,P<0.05),提示其对NOD小鼠产生免疫耐受有一定的作用,为研制乳酸菌口服疫苗防治T1DM的可行性进行了有益的探索。     

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.     

猪白细胞介素18成熟蛋白基因的克隆与表达

目的克隆猪白细胞介素18(pIL-18)成熟蛋白基因,并在植物乳杆菌(Lb.plantarum)NC8中进行表达。方法通过RT-PCR方法从猪脾脏细胞中扩增出pIL-18成熟蛋白基因,克隆到T载体pMD18-T后测序;将阳性基因片段克隆至大肠埃希菌-乳酸菌穿梭表达载体pSIP-409构建重组表达载体pSIP-409-IL-18,进行酶切和PCR鉴定;应用电穿孔技术将其转化至Lb.plantarum NC8中,经SppIP诱导表达后,进行SDS-PAGE及Western-blot分析。结果经测序,pIL-18成熟蛋白基因核苷酸长度为579 bp,编码193个核苷酸;酶切和PCR鉴定证明成功构建了重组表达载体pSIP-409-IL-18;SDS-PAGE及Western-blot分析表明重组菌表达了18 kD的融合蛋白,该重组蛋白可以与鼠抗猪IL-18多克隆抗体反应。结论成功克隆了pIL-18成熟蛋白基因,并获得有生物活性的pIL-18重组乳酸菌,为研制开发IL-18重组乳酸菌制剂奠定基础。     

乳酸菌应激反应及其在生产中的应用

适应性反应是乳酸菌应激保护作用的常见方式。当细胞处于多种环境胁迫时,由一种适应性反应表达所诱导的交互保护作用,对细胞的生存很有利。乳酸菌的蛋白质组研究目前仍处于开始阶段。基因组和转录组分析无疑将补充现有的蛋白质组和遗传学知识。充分了解应激反应的机制可以更好地理解适应性反应和交互保护作用的基础,更合理地开发乳酸菌在工业生产中的应用。     

Sugar utilization and its control in hyperthermophiles

Many hyperthermophilic microorganisms show heterotrophic growth on a variety of carbohydrates. There has been considerable fundamental and applied interest in the utilization of glucose and its α- and β-polymers by hyperthermophiles. While glycolysis by Bacteria at high temperatures shows conventional characteristics, it has been found that glucose catabolism by hyperthermophilic Archaea differs from the canonical glycolytic pathways, involves novel enzymes, and shows a unique control. This review addresses these aspects with specific attention to Pyrococcus furiosus, which is one of the best studied hyperthermophilic Archaea, has the capacity to grow on a variety of sugars including the marine β-(1,3)-linked glucose polymer laminarin, and has been found to contain three novel glycolytic enzymes, two ADP-dependent kinases, and a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase. Received: January 22, 1998 / Accepted: February 16, 1998     

Regulation of hdc expression and HDC activity by enological factors in lactic acid bacteria

Aims: The aim of this work was to study the influence of enological factors on the histidine decarboxylase gene (hdc) expression and on histidine decarboxylase enzyme (HDC) activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. Methods and Results: Cell extracts and whole cells were used. Glucose, fructose, malic acid and citric acid diminished the hdc expression. Ethanol did not increase hdc expression or activity in cells, but increased HDC activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and l -lactic acid, and sulphur dioxide (SO₂) had no effect on enzyme synthesis and activity. Bacterial species differ in the relative enzymatic activity but all the factors affected similarly to L. hilgardii, P. parvulus and O. oeni. Conclusions: The hdc gene expression was lowered by glucose, fructose, malic acid, and citric acid, whereas ethanol enhanced the HDC enzyme activity. The conditions that normally occur during malolactic fermentation and later on, could favour histamine production. SO₂ could prevent bacterial growth, but does not diminish the HDC enzyme activity. Significance and Impact of the Study: Information on hdc expression and HDC activity can contribute to the prevention of histamine formation during wine production and storage.     

产Ⅱ类细菌素乳酸菌群体感应及其应用

群体感应(quorum sensing,QS)是微生物通过感知与细胞密度相关的信号分子的浓度来调控基因表达的一种行为。许多产Ⅱ类细菌素乳酸菌通过自诱导肽介导的QS系统调控其细菌素的合成。本文综述了乳酸菌Ⅱ类细菌素合成的QS调控现象、调控机制、QS系统组分以及QS的应用。产Ⅱ类细菌素乳酸菌QS的研究,必将为揭示发酵调控机理、调控发酵过程提供新的研究平台,为食品级基因表达系统的开发提供新的选择。     

Antifungal properties of selected lactic acid bacteria and application in the biological control of ochratoxin A producing fungi during cocoa fermentation

Thirteen Lactic acid bacteria strains isolated from fermenting cocoa and seven reference strains were used in order to assess their antifungal properties towards three ochratoxin A (OTA) producing fungi (Aspergillus carbonarius, Aspergillus niger and Aspergillus ochraceus). Furthermore, two of the isolates strains (A19 and A21) identified as belonging to the genus of Pediococcus as well as Lactobacillus plantarum B4496, Lactobacillus brevis 207 and Lactobacillus sanfranciscensis BB12 showed interesting in vitro broad antifungal activities towards the three ochratoxin-producing fungi with inhibition percentages ranging from 15% to 66.7%. Treatment of cell-free supernatant at 100°C affected antifungal activity suggesting that the main compounds responsible for this activity were of proteic nature, and hence could be bacteriocins. Application of isolate A19 in cocoa fermentation as starter inhibited the growth of each of the OTA-producing species. At the end of fermentation in boxes inoculated with A19, A. niger was not detectable while A. carbonarius concentration was found to be 2 Log CFU/g of wet beans. The assessment of the ochratoxin produced during fermentation of cocoa inoculated with A. carbonarius indicated that the use of isolate A19 as starter could reduce their level of growth so as to have only a toxin production of 0.0012 ± 0.0005 μg/kg after 40 days of storage, while this was 2.45 ± 0.35 μg/kg of fermented and dried cocoa beans in the absence of A19. This work is a contribution for the application of biological control of OTA-producing fungi during cocoa production.     

Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.     

基因表达系列分析法(SAGE)的改进及其在植物功能基因组研究中的应用

基因表达系列分析方法(SAGE)是一种新的基因表达分析方法,与基因芯片技术一样具有高通量的特点,可测定特定组织的基因表达水平,在全基因组水平上同时定量检测数万个基因表达模式;可在未知目的基因的前提下,分析来自一个细胞的全部转录本信息;对已知或未知基因表达进行定性和定量分析.目前,虽然在疾病、发育、细胞凋亡、药物筛选等多个领域已有利用SAGE方法进行的研究,但该方法在植物功能基因组研究中的应用相对较少.本文主要综述了该方法在RNA用量、PCR循环次数、SAGE效能和可靠性、标签长度和未知标签分析等方面的改进及其在植物中构建SAGE文库、筛选新基因、基因表达图谱分析等方面的应用,从而为其在植物功能基因组研究中的进一步应用提供理论参考.     

Predicted highly expressed genes in Nocardia farcinica and the implication for its primary metabolism and nocardial virulence

Nocardia farcinica is a Gram positive, filamentous bacterium, and is considered an opportunistic pathogen. In this study, the highly expressed genes in N. farcinica were predicted using the codon adaptation index (CAI) as a numerical estimator of gene expressivity. Using ribosomal protein (RP) genes as references, the top ∼ ∼10% of the genes were predicted to be the predicted highly expressed (PHX) genes in N. farcinica using a CAI cutoff of greater than 0.73. Consistent with earlier analysis of Streptomyces genomes, most of the PHX genes in N. farcinica were involved in various ‘house-keeping’ functions important for cell growth. However, 15 genes putatively involved in nocardial virulence were predicted as PHX genes in N. farcinica, which included genes encoding four Mce proteins, cyclopropane fatty acid synthase which is involved in the modification of cell wall which may be important for nocardia virulence, polyketide synthase PKS13 for mycolic acid synthesis and a non-ribosomal peptide synthetase involved in biosynthesis of a mycobactin-related siderophore. In addition, multiple genes involved in defense against reactive oxygen species (ROS) produced by the phagocyte were predicted with high expressivity, which included alkylhydroperoxide reductase (ahpC), catalase (katG), superoxide dismutase (sodF), thioredoxin, thioredoxin reductase, glutathione peroxidase, and peptide methionine sulfoxide reductase, suggesting that combating against ROS is essential for survival of N. farcinica in host cells. The study also showed that the distribution of PHX genes in the N. farcinica circular chromosome was uneven, with more PHX genes located in the regions close to replication initiation site. The results provided the first estimates of global gene expression patterns in N.␣farcinica, which will be useful in guiding experimental design for further investigations. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Phenotypic, genotypic and technological characterization of predominant lactic acid bacteria in Pecorino cheese from central Italy

AIMS: Identification and biotyping of lactic acid bacteria (LAB) isolated from raw-milk Pecorino cheese manufactured in the Marche region (central Italy) for selection of suitable starter cultures or adjuncts. METHODS AND RESULTS: Preliminary characterization with morphological and biochemical assays were undertaken for 112 Gram-positive and catalase-negative isolates. Unequivocal identification of the isolates was obtained through restriction analysis of the amplified 16S rRNA gene and sequencing of 360-380 bp amplicons. Fifty-nine isolates belonging to LAB species generally recognized as safe and potentially utilized as starters or flavour-producing adjuncts were preselected and tested for their acidifying, proteolitic and autolytic activities. Fifty-five of these isolates were also subject to RAPD (randomly amplified polymorphic DNA) fingerprinting and unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis for the estimation of genotypic intra-species variation. As a result, in Pecorino cheese, a heterogeneous lactic acid bacteria population, which includes strains with metabolic characteristics of technological interest, was characterized. CONCLUSIONS: The polyphasic approach proposed allows the bacterial ecology of Pecorino cheese to be investigated and allows to assess the potential role of autochthonous LAB strains for the dairy industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The great economic importance of Pecorino cheese encouraged a deeper knowledge of its microbiota, which is known to influence the peculiar sensory properties of this cheese, also in view of its exploitation.     

Visinin-like peptide 1 in adrenal gland of the rat. Gene expression and its hormonal control

VSNL1 encodes the calcium-sensor protein visinin-like 1 and was identified previously as an upregulated gene in a sample set of aldosterone-producing adenomas. Recently, by means of microarray studies we demonstrated high expression of Vsnl1 gene in rat adrenal zona glomerulosa (ZG). Only scanty data are available on the role of this gene in adrenal function as well as on regulation of its expression by factors affecting adrenal cortex structure and function. Therefore we performed relevant studies aimed at clarifying some of the above issues. By Affymetrix^® Rat Gene 1.1 ST Array Strip, QPCR and immunohistochemistry we demonstrated that expression levels of Vsnl1 in the rat adrenal ZG are notably higher than in the fasciculata/reticularis zone. In QPCR assay this difference was approximately 10 times higher. Expression of this gene in the rat adrenal gland or adrenocortical cells was acutely down regulated by ACTH, while chronic administration of corticotrophin or dexamethasone did not change Vsnl1 mRNA levels. In enucleation-induced adrenocortical regeneration expression levels of both Vsnl1 and Cyp11b2 were notably lowered and positively correlated. Despite these findings, the physiological significance of adrenal Vsnl1 remains unclear, and requires further investigation     

梨火疫病拮抗菌筛选及温室防效测定

【背景】梨火疫病是由梨火疫病菌(Erwinia amylovora)引起的细菌性病害,对苹果、梨、山楂等40余属220多种植物危害极大。近年来,与中国毗邻的中亚多国相继暴发梨火疫病。梨火疫病菌成为国内特别是对新疆地区的特高风险有害生物,梨火疫病已被中国农业农村部列入《一类农作物病虫害名录》。【目的】从新疆本土材料以及实验室现有菌株中进行梨火疫病菌拮抗菌株的筛选,为梨火疫病的防控奠定基础。【方法】采用平板对峙法从新疆香梨种植区土壤及实验室现有菌株中分离、筛选具有显著拮抗效果的菌株,通过生理生化特性分析和16SrRNA基因测序等方法对菌株进行初步鉴定,并采用牛津杯扩散法对抑菌效果进行复筛。利用两年生盆栽杜梨苗对拮抗菌进行温室内保护型和治疗型防效测定。【结果】筛选获得11株具有显著拮抗作用的菌株,均为革兰氏阳性菌。其中9株为芽胞杆菌属(Bacillus)、2株为乳杆菌属(Lactobacillus)。平板对峙实验结果表明贝莱斯芽胞杆菌(Bacillus velezensis) JE7、植物乳杆菌(Lactobacillus plantarum) LP1和植物乳杆菌LP2拮抗作用较强,其次为贝莱斯芽胞杆菌JE4。生理特性实验结果表明:JE4、JE7可在NaCl浓度为1%-7%时生长,JE4、JE7在pH 4.0-9.0时生长,LP1可在NaCl浓度为1%-9%时生长,在pH 5.0-9.0时生长良好。温室防效测定结果显示,贝莱斯芽胞杆菌JE4防效最佳,可达到73%以上。【结论】筛选获得一批梨火疫病拮抗菌,其中部分菌株在温室内防效显著并具有较强的盐碱耐受能力。     

Characterization of soluble and putative membrane-bound UDP-glucuronic acid decarboxylase (OsUXS) isoforms in rice

Arabinoxylans in crop plants are the major sugar components of the cell walls, and UDP-xylose is a key substrate in the biosynthesis of xylans. In this study, the six putative UDP-D-glucuronic acid decarboxylase genes from rice (Oryza sativa UDP-xylose synthase; OsUXS) were cloned. Except for the soluble form of OsUXS3 (GenBank Accession No. \AB079064), the remaining five OsUXS enzymes contain a putative membrane-bound region. The six OsUXS genes were classified into three types by phylogenetic analysis and were expressed during the development of rice seeds. The HPLC retention times of the enzyme products and NMR data, indicate that the recombinant OsUXS2 enzyme catalyzes the conversion of UDP-D-glucuronic acid to UDP-D-xylose. Interestingly, the reactions catalyzed by the recombinant OsUXS2 and OsUXS3 enzymes were inhibited by NADP+, and accelerated by NADPH. The catalytic activities of the recombinant OsUXS2 and OsUXS3 enzymes were strongly inhibited by UDP, UTP, TDP, and TTP. The expression levels of OsUXS genes changed in different manners during the development of rice seeds, suggesting that each corresponding OsUXS enzyme plays a significant role in rice seed development at a certain stage. In the present study, we report that the UXS2-type enzyme of rice is not only characterized for the first time but also show significant findings involved in the gene expression of OsUXSs.     

Rhizosphere bacteria of maize with chitinolytic activity and its potential in the control of phytopathogenic fungi

The chitinase enzyme was identified in isolated bacteria of maize rhizosphere as well as its potential for the biological control of fungi associated at seeds of the same plant. The production of chitinase enzyme was found in the genera identified as Acinetobacter, Bacterium, Burkholderia, Paenibacillus, Pseudomonas, Rhizobium, Shewanella, Sphingomonas and Stenotrophomonas. Bacterial isolates with ability to degrade fungal mycelium from maize fungi as Fusarium and Alternaria among others, were detected. Bacterial chitinase activity and the presence of the chiA gene were determined. The inoculation of chitinolytic bacteria showed a positive effect in the control of fungi in maize seeds. The results support the potential use of chitinase enzyme producing bacteria on the control of phytopathogenic fungi.